The ability to introduce nucleic acids into cells and tissues has opened the door to new areas of bioscience research and therapy. A great deal of research has focused on viral and non-viral vectors to overcome the technical hurdles associated with gene delivery. Viral vectors commonly used to deliver exogenous genetic material to host cells include retrovirus, adenovirus and adeno-associated virus.
Retroviruses are enveloped viruses that contain a single-stranded RNA genome. The viral genome is small and uncomplicated, thereby facilitating manipulation and insertion of foreign genes. However, most retroviruses are able to integrate only into the genome of actively replicating target cells. Retroviral vectors are inefficient as far as cell transduction and are limited as to the size of genetic material that can be incorporated (Smith, Annu. Rev. Microbiol., 49, 807-835 (1995)).
Adenovirus is a non-enveloped virus containing linear double-stranded DNA and is associated with mild disease in humans. Adenovirus has the ability to infect a great number of cell types and can infect both dividing and non-dividing cells. However, adenovirus does not incorporate genetic material into the genome of the target cell, instead residing in episomal form. Adenoviral infection is also associated with the formation of an immune response. Therefore, adenovirus infection results in only transient expression of proteins.
Adeno-associated virus (AAV) is a parvovirus, which is dependent on co-infection with another virus for efficient infection of host cells. AAV is a single-stranded DNA virus, which is not associated with any human disease. The recombinant form of the virus (rAAV), which is used most often in in vivo research and clinical trials, has 96% of the viral genome deleted. rAAV is, therefore, incapable of producing the proteins required for replication and encapsidation. As such, rAAV requires both wild-type adenovirus and wild-type AAV or, in the alternative, recombinant plasmids comprising the required complementing genetic material to produce progeny. The use of recombinant AAV has an obvious safety advantage in that the probability of generating wild-type virus is low. Although administration of AAV may induce the production of anti-capsid antibodies, the deletion of the viral genome from rAAV lessens the immune response associated with viral infection, while increasing the amount of exogenous genetic material that can be inserted.
AAV offers prolonged transduction and expression. It has been suggested that this advantage of AAV may stem from the virus's reported ability to integrate into the host cell genome, although it has been demonstrated that AAV does not always so integrate. Despite the potential for prolonged expression, there are many limitations associated with AAV which hinder its use as a gene transfer vector. For example, while AAV can be produced in high titers, the vector is difficult to generate, due to lack of packaging cells and the need for helper virus. The size of genetic material to be introduced into AAV is limited to approximately 4.7 kb, which precludes many desirable therapeutic genes. In addition, the single-stranded AAV genome requires DNA synthesis to double-stranded DNA for a transgene to be expressed. This requirement has been thought to hinder transduction in non-dividing cells or cells with limited DNA synthesis. There also are conflicting reports regarding the ability of rAAV to integrate into target cells, particularly cells in vivo, to obtain long-term expression. (Xiao et al., Experimental Neurology, 114, 113-124 (1997)).
Viral-based methods of gene delivery often do not efficiently infect a broad range of cells or do not direct stable long-term transduction. In either case, repeated administration or large dosages are required to effect substantial, prolonged expression of genetic material. In addition, administration of virus induces an immune response that prevents effective repeated administration of the vectors. While it is possible to administer viral vectors displaying altered antigenic determinants or vary the types of vectors to elude the immune system, preparation and administration of the modified vectors places a significant burden on the researcher or physician.
Non-viral delivery methods, i.e., plasmids and liposome-DNA complexes, do not elicit the immune response associated with some viral vectors, thereby permitting repeated administration of large quantities of vector. However, the efficiency of non-viral mediated transduction is quite low. In situations where non-viral vectors transduce a significant number of cells, gene expression is not stable and diminishes quickly over time.
While introduction of genetic material into cells and tissues has been achieved, there has been little success in achieving prolonged and substantial expression. Therefore, there exists a need for a method of obtaining prolonged, substantially undiminished expression of exogenous nucleic acids in vivo. The present invention provides such a method. This and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.